Electrochemical cell

ABSTRACT

The present invention relates to electrochemical cells including a first working electrode  32 , a first counter electrode  34 , a second working electrode  36 , and a second counter electrode  38 , wherein the electrodes are spaced such that reaction products from the first counter electrode  34  arrive at the first working electrode  32 , and reaction products from the first and second counter electrodes  34, 38  do not reach the second working electrode  36 . Also provided is a method of using such electrochemical cells for determining the concentration of a reduced or oxidized form of a redox species with greater accuracy than can be obtained using an electrochemical cell having a single working and counter electrode.

This application is a 371 of PCT/US02/31289, filed on Oct. 01, 2002,which claims the benefit of U.S. provisional application No. 60/328,846,filed on Oct. 10, 2001.

FIELD OF THE INVENTION

The present invention relates to electrochemical cells including, afirst working electrode, a first counter electrode, a second workingelectrode and a second counter electrode, wherein the electrodes arespaced such that reaction products from the first counter electrodearrive at the first working electrode, and reaction products from thefirst and second counter electrodes do not reach the second workingelectrode. Also provided is a method of using such electrochemical cellsfor determining the concentration of a reduced or oxidized form of aredox species with greater accuracy than can be obtained using anelectrochemical cell having a single working and counter electrode.

BACKGROUND OF THE INVENTION

In amperometric electrochemistry the current flowing at the electrodecan be used as a measure of the concentration of electroactive speciesbeing reacted electrochemically at the working electrode. In coulometrythe current flowing at the electrode can be integrated over time to givea total amount of charge passed which yields a measure of the amount ofelectroactive material reacted at the working electrode. The currentflowing (or charge passed at any time) at the electrode depends upon therate of transfer of the electroactive species to the working electrode.When a significant concentration of electroactive species is situatedclose to the electrode and an electrical potential is applied to theelectrode sufficient to electrochemically react the electroactivespecies at the electrode/solution interface, initially a higher currentflows which diminishes with time. For an isolated and substantiallyplanar electrode, where the. potential applied to the electrode issufficient to react the electroactive species effectivelyinstantaneously upon arriving at the electrode and the transfer ofelectroactive species to the electrode is controlled by diffusion, thecurrent follows a curve known in the art as the Cottrell Equation.According to this equation the current varies inversely with the squareroot of time. This yields a current which decays with time as theelectroactive species that reacts at the electrode becomes depletedclose to the electrode and so electroactive species has to travel fromfurther and further away to reach the electrode as time progresses.

If, in addition to the electrochemical reaction of the electroactivespecies at the electrode, the electroactive species is generated closeto the working electrode by a chemical reaction, the form of the currentflowing at the electrode becomes complex. The electrode reaction tendsto decrease the concentration of electroactive species close to theworking electrode whereas the chemical reaction tends to increase theconcentration of the electroactive species in this region. The timedependent behavior of these two processes therefore mix and it can bedifficult to measure the chemical reaction kinetics from the currentflowing (or charge passed) at the electrode.

For this reason, in the published literature, the rates of chemicalreactions are not generally measured electrochemically except inspecialized applications using specialized equipment. An example of suchequipment is known in the art as a rotating ring/disc electrode. Thisapparatus is only applicable to relatively fast reaction kinetics andrequires that the electrode be rotated at a known controlled rate withwell-characterized liquid hydrodynamics.

SUMMARY OF THE INVENTION

An electrochemical cell and method of using such an electrochemical cellfor determining the concentration of a reduced or oxidized form of aredox species with greater accuracy than can be obtained using anelectrochemical cell having a single working and counter electrode isdesirable. The preferred embodiments provide such electrochemical cellsand methods.

In a first embodiment, a method for determining the concentration of areduced or oxidized form of a redox species is provided, the methodincluding the steps of: providing an electrochemical cell including afirst working electrode, a first counter electrode, a second workingelectrode and a second counter electrode; selecting the spacing betweenthe first working electrode and the first counter electrode so thatreaction products from the first counter electrode arrive at the firstworking electrode; selecting the spacing between the first workingelectrode and the second counter electrode so that a significant amountof reaction products from the second counter electrode do not arrive atthe first working electrode; selecting the spacing between the secondworking electrode and the second counter electrode so that a significantamount of reaction products from the second counter electrode do notarrive at the second working electrode; applying an electric potentialdifference between the first working electrode and the first counterelectrode; applying an electric potential difference between the secondworking electrode and second counter electrode; selecting the potentialof the first working electrode such that the rate of electro-oxidationof the reduced form or electro-reduction of the oxidized form of thespecies is diffusion controlled; selecting the potential of the secondworking electrode such that the rate of electro-oxidation of the reducedform or electro-reduction of the oxidized form of the species isdiffusion controlled; subtracting a current flowing between the secondworking electrode and the second counter electrode from a currentflowing between the first working electrode and the first counterelectrode, whereby a corrected current is obtained; and obtaining fromthe corrected current a value indicative of the concentration of thereduced form or the oxidized form of the species.

In one aspect of the first embodiment, the surface area of the firstworking electrode and a surface area of the second working electrode aresubstantially the same.

In another aspect of the first embodiment, the surface area of the firstworking electrode and a surface area of the second working electrode aredifferent, and the step of subtracting a current includes: determining acurrent flowing between the first working electrode and the firstcounter electrode; determining a current flowing between the secondworking electrode and the second counter electrode; normalizing thecurrent flowing between the first working electrode and the firstcounter electrode and the current flowing between the second workingelectrode and the second counter electrode to a same electrode surfacearea to yield a normalized current flowing between the first workingelectrode and the first counter electrode and a normalized currentflowing between the second working electrode and the second counterelectrode; and subtracting the normalized current flowing between thesecond working electrode and the second counter electrode from thenormalized current flowing between the first working electrode and thefirst counter electrode, whereby a corrected current is obtained.

In a further aspect of the first embodiment, the first working electrodeand the first counter electrode are separated by less than about 500 μm,or by less than about 200 μm. The second working electrode and thesecond counter electrode or the first working electrode and the secondcounter electrode are separated by more than about 500 μm, or by morethan about 1 mm.

In yet another aspect of the first embodiment, the redox species may bea mediator. When the redox species is a mediator, the concentration ofthe reduced or oxidized form of the mediator is indicative of theconcentration of an analyte and wherein a measure of the diffusioncoefficient of the reduced or oxidized form of the mediator isdetermined as a precursor to the determination of the concentration ofthe analyte.

In a further aspect of the first embodiment, the electrochemical celladditionally includes a separate reference electrode.

In yet another aspect of the first embodiment, the analyte may beglucose.

In a second embodiment, an electrochemical cell is provided including afirst working electrode, a first counter electrode, a second workingelectrode and a second counter electrode, the first working electrodebeing spaced from the first counter electrode by less than about 500 μm,the first working electrode being spaced from the second counterelectrode by more than about 500 μm, and the second working electrodebeing spaced from the second counter electrode by more than about 500μm.

In one aspect of the second embodiment, the first working electrode andthe first counter electrode and/or the second working electrode and thesecond counter electrode are facing one another or are in a side-by-sideconfiguration.

In another aspect of the second embodiment, the first working electrodeand the second working electrode are of substantially correspondingarea.

In a further aspect of the second embodiment, the electrochemical cellfurther includes a separate reference electrode.

In yet another aspect of the second embodiment, the electrochemical cellmay be a hollow electrochemical cell. The electrochemical cell can havean effective cell volume of less than 1.5 microliters.

In a third embodiment, an apparatus for determining the concentration ofa redox species in an electrochemical cell is provided including: anelectrochemical cell having a first working electrode, a first counterelectrode, a second working electrode and a second counter electrode,characterized in that the first working electrode is spaced from thefirst counter electrode by less than 500 μm, the first working electrodeis spaced from the second counter electrode by more than 500 μm, and thesecond working electrode is spaced from the second counter electrode bymore than 500 μm; means for applying an electric potential differencebetween the first working electrode and the first counter electrode; andmeans for applying an electric potential difference between the secondworking electrode and the second counter electrode.

In one aspect of the third embodiment, the apparatus may be a glucosemeter.

In a fourth embodiment, an electrochemical cell is provided including afirst working electrode, a first counter electrode, and a second workingelectrode, the first working electrode being spaced from the firstcounter electrode by less than about 500 μm, and the second workingelectrode being spaced from the first counter electrode by more thanabout 500 μm.

In a fifth embodiment, a method for determining the concentration of areduced or oxidized form of a redox species is provided, the methodincluding the steps of: providing an electrochemical cell including afirst working electrode, a counter electrode, and a second workingelectrode; selecting the spacing between the first working electrode andthe counter electrode so that reaction products from the counterelectrode arrive at the first working electrode; providing a redoxspecies, wherein at least a useful fraction of the redox speciesinitially present in the solution above the second working electrode hasbeen reduced or oxidized at the second working electrode; applying anelectric potential difference between the first working electrode andthe counter electrode; selecting the potential of the first workingelectrode such that the rate of electro-oxidation of the reduced form orelectro-reduction of the oxidized form of the species is diffusioncontrolled; determining a current flowing between the first workingelectrode and the counter electrode; and obtaining from the current avalue indicative of the concentration of the reduced form or theoxidized form of the species.

In one aspect of the fifth embodiment, a surface area of the firstworking electrode and a surface area of the second working electrode aresubstantially the same.

In another aspect of the fifth embodiment, a surface area of the firstworking electrode and a surface area of the second working electrode aresubstantially different.

In a sixth embodiment, a method for determining the concentration of areduced or oxidized form of a redox species is provided, the methodincluding the steps of: providing an electrochemical cell including afirst working electrode, a second working electrode, and a counterelectrode; selecting the spacing between the first working electrode andthe counter electrode so that reaction products from the counterelectrode arrive at the first working electrode; selecting the spacingbetween the second working electrode and the counter electrode so that asignificant amount of reaction products from the counter electrode donot arrive at the second working electrode; applying an electricpotential difference between the second working electrode and thecounter electrode whereby the second working electrode is substantiallycharged and whereby surface group reactions are substantially completed;interrupting the circuit between the second working electrode and thecounter electrode before a significant amount of the species is reactedat the second working electrode; applying an electric potentialdifference between the first working electrode and the counterelectrode; selecting the potential of the first working electrode suchthat the rate of electro-oxidation of the reduced form orelectro-reduction of the oxidized form of the species is diffusioncontrolled; determining a current flowing between the first workingelectrode and the counter electrode; and obtaining from the current avalue indicative of the concentration of the reduced form or theoxidized form of the species.

In a seventh embodiment, a method for determining the concentration of areduced or oxidized form of a redox species is provided, the methodincluding the steps of: providing an electrochemical cell including afirst working electrode, a second working electrode, and a counterelectrode; selecting the spacing between the first working electrode andthe counter electrode so that reaction products from the counterelectrode arrive at the first working electrode; selecting the spacingbetween the second working electrode and the counter electrode so that asignificant amount of reaction products from the counter electrode donot arrive at the second working electrode; applying an electricpotential difference between the second working electrode and thecounter electrode and between the first working electrode and thecounter electrode, whereby the second working electrode and firstworking electrode are substantially charged and whereby surface groupreactions are substantially completed; interrupting the circuit betweenthe second working electrode and the counter electrode before asignificant amount of the species is reacted at the second workingelectrode; applying an electric potential difference between the firstworking electrode and the counter electrode; selecting the potential ofthe first working electrode such that the rate of electro-oxidation ofthe reduced form or electro-reduction of the oxidized form of thespecies is diffusion controlled; determining a current flowing betweenthe first working electrode and the counter electrode; and obtainingfrom the current a value indicative of the concentration of the reducedform or the oxidized form of the species.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cross-section schematic of an electrochemical cell 10 ofa preferred embodiment with electrode surfaces in a parallel and opposedconfiguration.

FIG. 2 shows a cross-section schematic of an electrochemical cell 50 ofa preferred embodiment with electrodes in a side-by-side configuration.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following description and examples illustrate a preferred embodimentof the present invention in detail. Those of skill in the art willrecognize that there are numerous variations and modifications of thisinvention that are encompassed by its scope. Accordingly, thedescription of a preferred embodiment should not be deemed to limit thescope of the present invention.

It may be desirable when using electrochemical cells as amperometricsensors for the detection and quantification of analytes to be able todetect very low concentrations of the analyte. One of the limitations ofthe prior art in detecting low concentrations of an analyte can be thepresence of extraneous currents masking the current of interest. Some ofthese unwanted currents arise from the capacitive charging current ofthe electrode and electrical noise picked up from the environment. Thepreferred embodiments are directed towards a method for minimizing thecontribution of these currents to the overall signal, allowing forimproved detection of the analyte.

As is known in the prior art, electrodes in a two or three electrodeelectrochemical cell can be positioned such that the working electrodeis isolated from the counter electrode reactions and reaction productsor such that products of the counter electrode reaction diffuse to theworking electrode where they react. The former type of electrochemicalcell is well known in the prior art. The latter type of electrochemicalcell is discussed in U.S. Pat. Nos. 6,179,979 and 5,942,102.

These two electrode configurations vary in that in the isolated case,the counter electrode is positioned far enough away from the workingelectrode such that during the time the cell is being used, products ofelectrochemical reactions at the counter electrode do not reach theworking electrode. In practice, this is typically achieved by aseparation of the working electrode from the counter electrode by atleast a millimeter.

In the non-isolated configuration, the working electrode and the counterelectrode are placed close enough together such that products of theelectrochemical reactions at the counter electrode can diffuse to theworking electrode during the time the cell is being used. These reactionproducts can then react at the working electrode, giving a highercurrent than may be present in the isolated electrode case. In thenon-isolated configuration, the working electrode reactions can bedescribed as coupled to the counter electrode reactions.

Electrode Configurations

In a preferred embodiment, isolated working electrodes and workingelectrodes coupled to a counter electrode are combined in anelectrochemical cell to yield improved detection of low concentrationspecies. FIGS. 1 and 2 illustrate different electrode configurations inelectrochemical cells of preferred embodiments.

FIG. 1 shows a cross-section schematic of an electrochemical cell 10 ofa preferred embodiment. The exposed portions of electrically conductivelayers 12, 14, 16, 18 function as electrodes 32, 34, 36, 38 in the cell10. The electrically conductive layers 12, 14, 16, 18 are in contactwith layers 20, 22, 24, 26 of electrically resistive material. One ormore spacer layers (not illustrated) maintain the separation of theelectrodes 32, 34 to less than 500 μm. Either electrode 32 or electrode34 or electrode 36 or electrode 38 can be working electrodes, providedthat electrode 32 and 34 form one working and counter electrode pair andthat electrode 36 and electrode 38 form another working and counterelectrode pair. The thicknesses of layer 24 and layer 26 are such thatseparation between the closest edges of electrode 32 and electrode 36,and between the closest edges of electrode 34 and electrode 38 are alltypically greater than 500 μm, preferably greater than 1 mm. In anotherembodiment, the layer of electrically resistive material 20 or 22 andthe conductive layer 16 or 18 it supports may be substituted by a singlelayer of a suitable electrically conductive material (not illustrated),such as, for example, aluminum foil or a conducting polymer. For ease offabrication, in certain embodiments it may be desirable to completelycover one surface of one or more of the layers of electrically resistivematerial 20, 22, 24, 26 with an electrically conductive layer 12, 14,16, 18. Alternatively, in other embodiments it may be desirable to onlypartially cover the electrically resistive material 20, 22, 24, 26 withan electrically conductive layer 12, 14, 16, 18, for example, to save onmaterials costs if the electrode material comprises a noble metal. Forexample, in a cell 10 as illustrated in FIG. 1, the conductive layer 12may only cover the portion of the insulating layer 26 adjacent to thesample reservoir 28. The portion of the insulating layer 20 adjacent tolayer 26 is not covered. Other configurations of the electricallyconducting layer 12, 14, 16, 18 and its adjacent layer of electricallyresistive material 20, 22, 24, 26 will be apparent to one skilled in theart.

Another electrode configuration in an electrochemical cell 50 of apreferred embodiment is shown in FIG. 2. In this configuration, theelectrodes 52, 54, 56, 58 are all on the same plane. A spacer layer 60positioned over electrode 52 and electrode 54 is depicted in FIG. 2.When the electrochemical cell 50 is used in conjunction with a currentsubtraction method as described below, it may be preferred to omit thespacer layer 60. When the spacer layer 60 is omitted, the planardiffusion to electrode 54 more closely matches the planar diffusion toelectrode 58, resulting in a more accurate current subtraction.

When the electrochemical cell 50 is used in conjunction with a currentamplification method as described below, then it is preferred tomaintain the spacer layer above electrode 52 and electrode 54 so as toprovide a smaller volume of space 62 and a corresponding higheramplification factor than if the spacer layer 60 were not there. One ormore spacer layers (not illustrated) maintains the separation of theelectrodes 52, 54, 56, 58 from layer 64, thereby providing a samplereservoir 66 in the electrochemical cell 50. The distance between theclosest edges of electrode 52 and electrode 54 is less than 500 μm,preferably less than about 450, 400, 350, 300, or 250 μm, morepreferably less than about 200, 150, or 100 μm, and most preferably lessthan about 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 5 or 1 μm. Theseparation between the closest edges of electrode 52 and electrode 58,and between the closest edges of electrode 54 and electrode 58 aretypically greater than about 500 μm, preferably greater than about 550,600, 650, 700, 750, 800, 850, 900, or 950 μm, and most preferablygreater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, or 50 mm. However, anysuitable spacing, including spacings less than about 500 μm, may besuitable so long as a significant amount of the products of reaction atthe first electrode do not reach the second electrode. In this context asignificant amount of reaction products is an amount sufficient to giverise to an extra amount of current at the second electrode that is largeenough that it effects the practical utility of the methods of use ofthe cells to achieve the desired outcomes. In certain embodiments, itmay be preferred to omit any spacer layers and layer 64, therebyproviding an electrochemical cell including the electrodes 52, 54, 56,58 on a single layer of electrically resistive material 68. Thisembodiment may be preferred when sample sizes are sufficient such thatthe layer 68 and electrodes 52, 54, 56, 58 may be immersed in thesample, or a sufficient layer of sample may be applied to electrodes 52,54, 56, 58.

As will be apparent to one skilled in the art, different electrodeconfigurations maintaining the appropriate spacing between electrodes52, 54, 56, 58 may be preferred in various embodiments. For example, theelectrochemical cell 50 illustrated in FIG. 2 may be modified by placingone or both of electrodes 56 and 58 on layer 64 instead of layer 68.Alternatively, one or both of electrodes 52 and 54 may be placed onlayer 64 or 60 instead of layer 68. If only one of electrodes 52 and 54is placed on layer 68, layers 64 and 68 or layers 60 and 68 are placedsufficiently close such that the spacing between the closest edges orsurfaces of electrodes 52 and 54 is maintained at less than 500 μm,preferably less than about 450, 400, 350, 300, or 250 μm, morepreferably less than about 200, 150, or 100 μm, and most preferably lessthan about 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 5 or 1 μm. Inanother embodiment, an additional layer (not illustrated) is placed onlayer 68, and one or both of electrode 52 and electrode 54 may be placedon the additional layer.

Fabricating the Electrochemical Cell

The electrochemical cell of certain embodiments is disposable anddesigned for use in a single experiment. In preferred embodiments, theelectrochemical cell may be fabricated using methods similar to thosedisclosed in U.S. Pat. No. 5,942,102. In one method of a preferredembodiment for preparing an electrochemical cell 10 as illustrated inFIG. 1, the layers of electrically resistive material 20, 22, 24, or 26are polyester sheets having a sputter coating of palladium as theelectrically conductive layer 12, 14, 16, or 18, the portion remainingexposed after fabrication forming the electrodes 32, 34, 36, or 38.

As will be recognized by one skilled in the art, the layers ofelectrically conductive material 12, 14, 16, 18 and layers ofelectrically resistive material 20, 22, 24, 26 may be independentlyselected as desired, for example, for ease of fabrication, for reducingmaterials costs, or to achieve other desirable attributes of the cell 10or fabrication process. Likewise, the layers of electrically conductivematerial 12, 14, 16, 18 may be applied to the layers of electricallyresistive material 20, 22, 24, 26 in any suitable pattern, for example,a pattern that only partially covers the electrically resistive layer20, 22, 24, or 26.

Once the electrically conductive materials are coated on or otherwiseadhered to the corresponding electrically resistive layers 20, 22, 24,or 26, the covered layers 40, 42 may then be adhered to each other toform an electrode bearing layer 48. In the electrochemical cell of FIG.1, covered layer 40 is adhered to covered layer 42 with the conductivelayer 16 adjacent to the electrically resistive layer 26 of coveredlayer 42. Covered layers 44 and 46 are likewise adhered to form anelectrode-bearing layer 49.

In preferred embodiments, various layers in the cell may be adheredusing a suitable adhesive. Suitable adhesives include, for example, heatactivated adhesives, pressure sensitive adhesives, heat cured adhesives,chemically cured adhesives, hot melt adhesives, hot flow adhesives, andthe like. Pressure sensitive adhesives are preferred for use in certainembodiments where simplification of fabrication is desired. However, inother embodiments the tackiness of pressure sensitive adhesives mayresult in fabrication tool gumming or product tackiness. In suchembodiments, heat or chemically cured adhesives are generally preferred.Especially preferred are the heat-activated and heat-cured adhesives,which can be conveniently activated at the appropriate time.

In certain embodiments, it may be preferred to use a hot melt adhesive.A hot melt adhesive is a solvent-free thermoplastic material that issolid at room temperature and is applied in molten form to a surface towhich it adheres when cooled to a temperature below its melting point.Hot melt adhesives are available in a variety of chemistries over arange of melting points. The hot melt adhesive can be in the form of aweb, nonwoven material woven material, powder, solution, or any othersuitable form. Polyester hot melt adhesives may be preferred for certainembodiments. Such adhesives (available, for example, from Bostik Corp.of Middleton, Mass.) are linear saturated polyester hot melts exhibitingmelting points from 65° C. up to 220° C. and range from completelyamorphous to highly crystalline in nature. Polyamide (nylon) hot meltadhesives, also available from Bostik, may also be preferred, includingboth dimer-acid and nylon-type polyamide adhesives. Suitable hot meltadhesive chemistries include EVA, polyethylene, and polypropylene.

Alternatively, in certain other embodiments it may be preferred to uselamination techniques to bond certain layers together. Suitablelamination techniques are described in copending application Ser. No.09/694,120 filed Oct. 20, 2000 and entitled “Laminates of AsymmetricMembranes.” The layers to be laminated are placed adjacent to each otherand heat is applied, whereby a bond between the layers is formed.Pressure may also be applied to aid in forming the bond. Laminationmethods may be preferred to bond any two materials capable of forming abond under application of heat and/or pressure. Lamination is preferredto form a bond between two suitable polymeric materials.

The electrode bearing layers 48 and 49 are then fixed in position withthe electrodes 32 and 34 facing electrodes 36 and 38. This is typicallyaccomplished by adhering one or more shaped spacer layers (notillustrated) between the electrode bearing layers 48 and 49. The spacerlayer is shaped so as to provide sample reservoirs 28 and 29 between theelectrode bearing layers 48 and 49. The spacer layer may be in the formof a sheet of electrically resistive material with a portion of thesheet removed to form the sample reservoirs 28 and 29, for example, acircular portion centered in the middle of the sheet, or a portionremoved along one edge of the sheet. The spacer layer may also includetwo or more shaped portions placed adjacent to each other with a spacebetween, the space providing entry of sample into sample reservoirs 28and 29 and the reservoirs themselves 28 and 29. Instead of a rigid orflexible sheet of material, a layer of electrically resistive adhesivemay be preferred as the spacer. In such an embodiment, the adhesive isapplied to the electrode side of an electrode bearing layer 48 or 49,then the other electrode bearing layer 49 or 48 is placed atop theadhesive layer and a bond is formed, for example, by pressure, curing,heat, or other suitable means.

In a preferred embodiment, the spacer layer is a sheet of electricallyresistive material pierced by a circular aperture and adhered by anadhesive to the electrode bearing layers 48 and 49. The circularaperture is preferably centered along the edge of electrode 32 adjacentto electrode 38 (or the edge of electrode 34 adjacent to electrode 38).There is thereby defined a cell 10 having a cylindrical side wall closedon end by electrode bearing layer 48 and on the other side by electrodebearing layer 49. The assembly is notched to provide for sample to beadmitted to the cell 10 or to be drawn in by wicking or capillary actionand to allow air to escape. The electrode layers 32, 34, 36, 38 areconnected with suitable electrical connections or formations wherebypotentials may be applied and currents measured.

In another preferred embodiment, the spacer is formed by applying apattern of adhesive to one or both of the electrode bearing layers 48,49. This method may be preferred where ease of fabrication and reductionin material costs are desired.

Suitable electrically resistive materials which may be preferred asspacer layers, as supports for electrode layers, or in other layers inthe cell, include, for example, materials such as polyesters,polystyrenes, polycarbonates, polyolefins, polyethylene terephthalate,glasses, ceramics, mixtures and/or combinations thereof, and the like.Examples of electrically resistive adhesives suitable for use as spacerlayers include, but are not limited to, polyacrylates,polymethacrylates, polyurethanes, and sulfonated polyesters.

In embodiments wherein the spacer is a sheet of electrically resistivematerial with a portion removed to form the sample reservoirs 28 and 29,one electrode bearing layer 48 or 49 is mounted on one side of thesheet, extending over the aperture and forming an end wall. Theelectrode-bearing layer 48 or 49 may be adhered to the spacer sheet, forexample, by an adhesive. Multiple spacer sheets may be adhered to eachother so as to form a spacer that conforms to the stepped surfaces ofthe electrode bearing layers 48 and 49. A deformable adhesive may alsobe preferred as the spacer, the adhesive conforming to the contours ofthe electrode bearing layers 48 and 49. In a preferred embodiment, theoverall shape of the combined sample reservoirs 28 and 29 is circular,however other shapes, for example, square, rectangular, polygonal, oval,ellipsoidal, irregular, or others, may be preferred for certainembodiments.

The second electrode bearing layer 49 or 48 is then mounted on theopposite side of the spacer, also extending over the aperture, so as toform a second end wall. Electrodes 32 and 34 are typically spaced lessthan about 500 μm apart, preferably less than about 450, 400, 350, 300,or 250 μm apart, more preferably less than about 200, 150, or 100 μmapart, and most preferably less than about 90, 80, 70, 60, 50, 40, 30,25, 20, 15, 10, 5 or 1 μm. A second aperture or ingress is then providedfor liquid to enter the cell 10. Such an ingress can be provided byforming a notch along one edge of the device, which extends through theelectrode bearing layers 48 and 49 and aperture. The electrode bearinglayers 48 and 49 are provided with connections allowing the electrodesto be placed in a measuring circuit.

As will be recognized by one skilled in the art, the techniquesdescribed above for fabricating an electrochemical cell as illustratedin FIG. 1 may be modified to fabricate an electrochemical cell asillustrated in FIG. 2.

Chemicals for use in the cell, such as redox reagents, lysing agents,buffers, inert salts, and other substances, may be supported on the cellelectrodes or walls, on one or more independent supports containedwithin cell, or may be self supporting. If the chemicals are to besupported on the cell electrodes or walls, the chemicals may be appliedby use of application techniques well known in the art, such as ink jetprinting, screen printing, lithography, ultrasonic spraying, slotcoating, gravure printing, and the like. Suitable independent supportsmay include, but are not limited to, meshes, nonwoven sheets, fibrousfillers, macroporous membranes, and sintered powders. The chemicals foruse in the cell may be supported on or contained within a support.

In a preferred embodiment, the preferred materials within the cell aswell as the materials from which the cell is constructed are in a formamenable to mass production, and the cells themselves are designed for asingle experiment then disposed of. A disposable cell is one that isinexpensive enough to produce that it is economically acceptable onlyfor a single test. A disposable cell is one that may conveniently onlybe used for a single test, namely, steps such as washing and/orreloading of reagents may need to be taken to process the cell after asingle use to render it suitable for a subsequent use.

Economically acceptable in this context means that the perceived valueof the result of the test to the user is the same or greater than thecost of the cell to purchase and use, the cell purchase price being setby the cost of supplying the cell to the user plus an appropriate markup. For many applications, cells having relatively low materials costsand simple fabrication processes are preferred. For example, theelectrode materials of the cells may be inexpensive, such as carbon, ormay be present in sufficiently small amounts such that expensivematerials may be preferred. Screen printing carbon or silver ink is aprocess suitable for forming electrodes with relatively inexpensivematerials. However, if it is desired to use electrode materials such asplatinum, palladium, gold, or iridium, methods with better materialutilization, such as sputtering or evaporative vapor coating, arepreferred as they may yield extremely thin films. The substratematerials for the disposable cells are also preferably inexpensive.Examples of such inexpensive materials are polymers such aspolyvinylchloride, polyimide, polyester and coated papers and cardboard.

Cell assembly methods are preferably amenable to mass production. Thesemethods include fabricating multiple cells on cards and separating thecard into individual strips subsequent to the main assembly steps, andweb fabrication where the cells are produced on a continuous web, whichis subsequently separated into individual strips. Card processes aremost suitable when close spatial registration of multiple features isdesired for the fabrication and/or when stiff cell substrate materialsare preferred. Web processes are most suitable when the down webregistration of features is not as critical and flexible webs may bepreferred.

A convenient single use for the disposable cell is desirable so thatusers are not tempted to try to reuse the cell and possibly obtain aninaccurate test result. Single use of the cell may be stated in userinstructions accompanying the cell. More preferably, in certainembodiments where a single use is desirable the cell may be fabricatedsuch that using the cell more than once is difficult or not possible.This may be accomplished, for example, by including reagents that arewashed away or consumed during the first test and so are not functionalin a second test. Alternatively, the signal of the test may be examinedfor indications that reagents in the cell have already reacted, such asan abnormally high initial signal, and the test aborted. Another methodincludes providing a means for breaking electrical connections in thecell after the first test in a cell has been completed.

The Electrodes

In a preferred embodiment wherein the electrochemical cell detects thepresence and/or amount of analyte in the sample, or a substanceindicative of the presence and/or amount of analyte present in thesample, at least one of the electrodes in the cell is a workingelectrode. When the potential of the working electrode is indicative ofthe level of analyte (such as in a potentiometric sensor) a secondelectrode acting as reference electrode is present which acts to providea reference potential.

In the case of an amperometric sensor wherein the working electrodecurrent is indicative of the level of an analyte, such as glucose, atleast one other electrode is preferably present which functions as acounter electrode to complete the electrical circuit. This secondelectrode may also function as a reference electrode. Alternatively, aseparate electrode may perform the function of a reference electrode.

Materials suitable for the working, counter, and reference electrodesare compatible with any reagents or substances present in the device.Compatible materials do not substantially react chemically with othersubstances present in the cell. Examples of such suitable materials mayinclude, but are not limited to, carbon, carbon and an organic binder,platinum, palladium, carbon, indium oxide, tin oxide, mixed indium/tinoxides, gold, silver, iridium, and mixtures thereof. These materials maybe formed into electrode structures by any suitable method, for example,by sputtering, vapor coating, screen printing, thermal evaporation,gravure printing, slot coating or lithography. In preferred embodiments,the material is sputtered or screen-printed to form the electrodestructures.

Non-limiting examples of materials preferred for use in referenceelectrodes include metal/metal salt systems such as silver in contactwith silver chloride, silver bromide or silver iodide, and mercury incontact mercurous chloride or mercurous sulfate. The metal may bedeposited by any suitable method and then brought into contact with theappropriate metal salt. Suitable methods include, for example,electrolysis in a suitable salt solution or chemical oxidation. Suchmetal/metal salt systems provide better potential control inpotentiometric measurement methods than do single metal componentsystems. In a preferred embodiment, the metal/metal salt electrodesystems are preferred as a separate reference electrode in anamperometric sensor.

The Lysing Agent

In certain embodiments, it may be desired to include one or more lysingagents in the electrochemical cell. Suitable lysing agents includedetergents, both ionic and non-ionic, proteolytic enzymes, and lipases.Suitable ionic detergents include, for example, sodium dodecyl sulfateand cetyl trimethylammonium bromide. Non-limiting examples ofproteolytic enzymes include trypsin, chymotrypsin, pepsin, papain, andPronase E, a very active enzyme having broad specificity. Nonionicsurfactants suitable for use include, for example, ethoxylatedoctylphenols, including the Triton X Series available from Rohm & Haasof Philadelphia, Pa. In a preferred embodiment, saponins, namely, plantglycosides that foam in water, are preferred as the lysing agent.

The Redox Reagent

Redox reagents may also be included in the electrochemical cell inpreferred embodiments. Preferred redox reagents for use inelectrochemical cells for measuring glucose in blood include those whichare capable of oxidizing the reduced form of enzymes that are capable ofselectively oxidizing glucose. Examples of suitable enzymes include, butare not limited to, glucose oxidase dehydrogenase, PQQ dependent glucosedehydrogenase, and NAD dependent glucose dehydrogenase. Examples ofredox reagents suitable for use in analyzing glucose include, but arenot limited, to salts of ferricyanide, dichromate, vanadium oxides,permanganate, and electroactive organometallic complexes. Organic redoxreagents such as dichlorophenolindophenol, and quinones are alsosuitable. In a preferred embodiment, the redox reagent for analyzingglucose is ferricyanide.

The Buffer

Optionally, a buffer may be present along with a redox reagent in driedform in the electrochemical cell. If a buffer is present, it is presentin an amount such that the resulting pH level is suitable for adjustingthe oxidizing potential of the redox reagent to a level suitable foroxidizing, for example, glucose but not other species that it is notdesired to detect. The buffer is present in a sufficient amount so as tosubstantially maintain the pH of the sample at the desired level duringthe test. Examples of suitable buffers include phosphates, carbonates,alkali metal salts of mellitic acid, and alkali metal salts of citricacid. The choice of buffer may depend, amongst other factors, on thedesired pH. The buffer is selected so as not to react with the redoxreagent.

Inert Salts

Inert salts preferred for use in various embodiments include salts thatdissociate to form ions in the sample to be analyzed, but do not reactwith any of the redox reagents or other substances in the sample or inthe cell, including with the cell electrodes. Examples of suitable inertsalts include, but are not limited to, alkali metal chlorides, nitrates,sulfates, and phosphates.

Other Substances Present within the Cell

In addition to redox reagents and buffers, other substances may also bepresent within the electrochemical cell. Such substances include, forexample, viscosity enhancers and low molecular weight polymers.Hydrophilic substances may also be contained within the cell, such aspolyethylene glycol, polyacrylic acid, dextran, and surfactants such asthose marketed by Rohm & Haas Company of Philadelphia, Pa., under thetrade name Triton™, or by ICI Americas Inc. of Wilmington, Del., underthe trade name Tween™. Such substances may enhance the fill rate of thecell, provide a more stable measurement, and inhibit evaporation insmall volume samples.

Electrical Circuit

The electrically conductive layers are preferably connected toelectrical circuits capable of applying potentials between theelectrodes and measuring the resulting currents, for example, meters.Any suitable means for connecting an electrically conductive layer to anelectrical circuit may be preferred, including, but not limited to, atongue plug, a set of connection pins that are brought down on top ofthe strip or up from below the strip, and the like. The connection areasare not illustrated in FIG. 1. Suitable meters may include one or moreof a power source, circuitry for applying controlled potentials orcurrents, a microprocessor control device, computer, or data storagedevice, a display device, an audible alarm device, or other devices orcomponents as are known in the art. The meter may also be capable ofbeing interfaced to a computer or data storage device. For example, atypical meter may be a hand-held device that is powered by a battery,controlled by an on-board microprocessor, and contains circuitry forapplying predetermined potentials or currents between, for example,strip electrode connection pins and circuitry such as ananalog-to-digital converter. In this embodiment, the analog signal fromthe strip may be converted to a digital signal that can be analyzedand/or stored by a microprocessor. The meter may also contain a displaysuch as a Liquid Crystal Display and suitable associated circuitry todisplay the result of the test to the user. In an alternativeembodiment, the meter may incorporate specialized circuitry, such aspotential application and signal acquisition circuitry. Such specializedcircuitry may be incorporated in a separate module that may beinterfaced with a generic computing device, such as a hand-held computeror other type of computer. In such an embodiment, the generic device mayperform the control, analysis, data storage, and/or display functions.Such an embodiment allows for a less expensive meter to be producedbecause the generic computing device may be preferred for many functionsand as such is not considered as part of the cost of the electrochemicalmeasurement system. In either of these meter embodiments, the meter orgeneric computing device may be capable of communication with externaldevices such as local computer networks or the Internet to facilitatethe distribution of test results and the provision of system upgrades tothe user.

Obtaining Electrochemical Measurements

An electrochemical cell as shown in FIG. 1 or FIG. 2 may be used toprovide improved analyte detection. However, for purposes ofillustration, the methods of preferred embodiments are discussed inregard to the electrochemical cell 10 of FIG. 1 wherein electrode 34 andelectrode 38 are set as working electrodes and electrode 36 andelectrode 32 as counter electrodes. The analyte in this context can bethe actual specie(s) of interest in the sample or can be products ofchemical reactions with the specie(s) of interest. Electrodes 32 and 34are spaced closely enough such that the products of electrochemicalreactions at electrode 32 diffuse to and react at electrode 34 duringthe time of the test. This spacing is typically less than about 500 μm,preferably less than about 450, 400, 350, 300, or 250 μm, morepreferably less than about 200, 150, or 100 μm, and most preferably lessthan about 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 5 or 1 μm.Electrodes 36 and 38, however, are spaced far enough apart such that theproducts of reaction at electrode 36 do not reach electrode 38 duringthe test. This space is typically greater than about 500 μm, preferablygreater than about 550, 600, 650, 700, 750, 800, 850, 900, or 950 μm andmost preferably greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, or50 mm. In any case, the gap between electrode 36 and electrode 38 istypically substantially larger than the gap between electrode 32 andelectrode 34.

When solution containing the analyte fills the spaces 29 and 28, apotential is applied between electrode 32 and electrode 34 by a firstexternal circuit and between electrode 36 and electrode 38 by a secondexternal circuit. These potentials are of a polarity such that theanalyte is electrochemically reacted at electrode 34 and electrode 38and of sufficient size such that the rate of electrochemical reaction islimited by the rate of mass transport of analyte to electrode 34 orelectrode 38. As the potentials continue to be applied products of theelectrochemical reactions at electrode 32 diffuse to electrode 34 andare reacted, however, there is no time for any significant amount of theproducts of the reactions at electrode 36 to reach electrode 38. Bysubtracting the current flowing between electrode 36 and electrode 38from that flowing between electrode 32 and electrode 34, a currentversus time signal can be obtained which is due only to the reaction atelectrode 34 of the products of the electrochemical reactions atelectrode 32. In order to obtain this current accurately, electrode 34and electrode 38 either have to be of the same area, or the separatecurrents normalized by their respective working electrode areas prior tothe current subtraction.

The advantage of obtaining the current only due to the reaction ofcounter electrode products at the working electrode is thatcontributions from other extraneous currents are eliminated. Theseextraneous currents include currents due to reaction of electrodesurface groups, currents due to the oxidation or reduction of adsorbedspecies, and the electrode charging current, that is, the current thatflows to polarize the electrode/solution double layer to the potentialthat is being applied across the interface by the external circuit.These currents flow at short times and limit the shortness of time atwhich current related to the analyte can be obtained with any certainty.By using this method to eliminate the extraneous current contributions,the current signal at shorter times can be used to obtain informationabout the analyte with increased certainty. It is desirable to be ableto use the current signal at shorter times as it allows electrode 32 andelectrode 34 to be placed closer together than may otherwise bepractical. By placing electrode 32 and electrode 34 closer, the productsfrom the reaction at electrode 32 reach electrode 34 faster and inhigher amount. This increases the current signal and shortens the timeperiod over which the currents are monitored to obtain the desiredanalyte information.

In this method of a preferred embodiment, electrodes 32 and 34 form onecircuit with a power supply to apply a suitable potential betweenelectrodes 32 and 34. A second circuit, separate from the first, isformed between electrodes 36 and 38 and a power supply such that thecurrent flowing between electrodes 32 and 34 and the current flowingbetween electrodes 36 and 38 can be measured separately. Alternatively,rather than measuring the currents separately, the two currents can besubtracted electronically and the resulting subtracted current measured.

In a second method of a preferred embodiment, an electrode arrangementcan be used to effectively amplify the current signal arising fromreaction of the analyte. In this method, electrode 32 is used as acounter electrode for both electrode 34 and electrode 38 during at leasta portion of the test. A reagent is dried or otherwise deposited withinthe space between electrode 32 and 34, the reagent including a mediatorthat is electrochemically reversible and preferably also reactschemically with the analyte of interest to produce a reacted mediator,wherein the reacted mediator is capable of reacting electrochemically atelectrode 34 and being electrochemically generated at electrode 32 frommediator. The reagent deposited within space 28 may contain a mediatoror, when the analyte is capable of reacting directly at the electrode38, may not contain a mediator.

During a test, potentials are applied such that the analyte and/or themediator that has chemically reacted with the analyte electrochemicallyreact at electrode 34 and 38. The counter electrode used to complete thecircuit for both electrode 34 and electrode 38 in this method of use iselectrode 32. Electrons gathered from reactions with the analyte orreacted mediator at electrode 38 leads to an equal amount of reactedmediator being produced at 32. This reacted mediator can then travel toelectrode 34 and react to be returned to mediator. In this way thecurrent arising from the analyte or reacted mediator in the volume ofsolution in space 28 is used to produce a corresponding amount ofreacted mediator in the volume of solution in space 29, thus effectivelyconcentrating a specie related to the analyte from space 28 into space29 producing an enhanced current signal from the analyte. Due to thediffusion distances involved, the reacted mediator in space 29 remainssubstantially in space 29 during the test. To ensure that this is thecase, it is preferred to have the length of space 29 longer than thedistance between electrode 36 and electrode 38. In this case, in thetime it takes for mediator to diffuse from electrode 36 to electrode 38,only a small fraction of the material in space 29 diffuses into space28.

By way of an example of this method, if the area of electrode 38 is tentimes that of electrode 34 and the thickness of space 28 is ten timesthe thickness of space 29, then the concentration of the reactedmediator in space 29 is up to 101 times that present than if justelectrode 32 and electrode 34 are used. In this example, therefore, thedetection limit of the analyte is lowered by up to 101 times. Forexample, if the concentration of analyte or reacted mediator in thesolution filling spaces 29 and 28 was originally X, then aftersubstantially all of the analyte or reacted mediator in the solutionabove electrode 38 has been electrochemically reacted at electrode 38,that number of moles of analyte or reacted mediator has produced acorresponding number of moles of reacted mediator in the space 29. Sincein this example the volume of solution above electrode 38 is 100 timesthe volume of the space 29 the concentration of reacted mediator inspace 29 is now X+100·X, the original amount in the space 29 plus 100times the original amount due the reactions at electrode 38. Note thatit is not necessary to react all the analyte or reacted mediator in thesolution above electrode 38 for this method to have utility. In somecases, for instance where it is desirable to sacrifice some signalamplification for a shorter test time, only a fraction of the analyte orreacted mediator in the solution above electrode 38 is reacted, as longas the fraction of species reacted is useful in that it is sufficient toobtain a useful signal amplification.

Optionally, in order to further reduce electrical noise, after thedesired fraction (typically substantially all) of the analyte or reactedmediator have been electrochemically reacted at electrode 38, thecircuit between electrode 32 and electrode 38 can be disconnected,leaving just electrode 32 and electrode 34 with a potential betweenthem. The current flowing between electrode 32 and electrode 34 can thenbe monitored to determine the concentration of reacted mediator in space29, which is related in a known way to the original analyteconcentration. This procedure reduces electrical noise during theconcentration determination as noise generated from electrode 38 iseliminated. The time at which the circuit between electrode 32 andelectrode 38 is disconnected can, for example, be determined by settinga threshold current between electrode 32 and electrode 38 below whichthe disconnection occurs. Note that in this method of measuring current,the second counter electrode 36 is not necessary and so can be omitted.

A further optional method for reducing electrical noise due to electrodecharging and other extraneous currents is to use electrode 36 as thecounter electrode for electrode 38 during the electrode charging phaseimmediately after the potential has been applied between electrode 36and electrode 38. After electrode 38 is polarized to the correctpotential, the counter electrode for electrode 38 can be switched to beelectrode 32. The time at which the counter electrode is switched can,for example, be set at a fixed time at which it is known that theelectrode charging and surface group reactions are substantially overbut before a substantial amount of the analyte or reacted mediator hasreacted at electrode 38. If electrode 36 and electrode 38 are ofsubstantially equivalent area then the charging current does not lead toany substantial amount of additional reacted mediator being formed dueto the charging process.

A further optional method for reducing noise due to electrode chargingand other extraneous currents is to use electrode 36 as the counterelectrode for electrodes 34 and 38 during the electrode charging phaseimmediately after the potential has been applied to electrodes 34 and38. After electrodes 34 and 38 are polarized to the correct potentialand after the electrochemical reaction of some or all of the surface andadsorbed groups or reacted mediator present in any dried reagent layersadjacent to electrode 34, the counter electrode for electrodes 34 and 38can be switched to electrode 36. As above, the time of the switching thecounter electrode can be at a fixed time. This procedure allows for theeffect of extraneous reacted mediator or other electrochemicallyreactive species to be lessened or eliminated. In these two options, thesecond counter electrode 36 is present.

In the above-mentioned methods, it is desirable that anelectrochemically inert soluble salt, at a concentration substantiallyhigher than the analyte, also be present in the solution filling thecell, either derived from the sample itself or from reagents depositedinto the cell. This inert salt serves to carry electrical current in thesolution between space 29 and space 28 when electrode 32 is used as thecounter electrode for electrode 38, minimizing the loss of reactedmediator from space 29 due to electromigration.

The above description provides several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention provided herein. Consequently, it is not intended that thisinvention be limited to the specific embodiments provided herein, butthat it cover all modifications and alternatives coming within the truescope and spirit of the invention as embodied in the attached claims.

1. A method for determining a concentration of a reduced or oxidizedform of a redox species, the method comprising the steps of: a)providing an electrochemical cell comprising a first working electrode,a first counter electrode, a second working electrode and a secondcounter electrode; b) selecting a spacing between the first workingelectrode and the first counter electrode such that reaction productsfrom the first counter electrode arrive at the first working electrode;c) selecting a spacing between the first working electrode and thesecond counter electrode such that a significant amount of reactionproducts from the second counter electrode do not arrive at the firstworking electrode; d) selecting a spacing between the second workingelectrode and the second counter electrode such that a significantamount of reaction products from the second counter electrode do notarrive at the second working electrode; e) applying an electricpotential difference between the first working electrode and the firstcounter electrode; f) applying an electric potential difference betweenthe second working electrode and second counter electrode; g) selectinga potential of the first working electrode such that a rate ofelectro-oxidation of a reduced form of a redox species or a rate ofelectro-reduction of an oxidized form of a redox species is diffusioncontrolled; h) selecting a potential of the second working electrodesuch that a rate of electro-oxidation of the reduced form of a redoxspecies or a rate of electro-reduction of an oxidized form of a redoxspecies is diffusion controlled; i) subtracting a current flowingbetween the second working electrode and the second counter electrodefrom a current flowing between the first working electrode and the firstcounter electrode, whereby a corrected current is obtained; and j)obtaining from the corrected current a value indicative of theconcentration of the reduced form of the redox species or the oxidizedform of the redox species.
 2. The method according to claim 1, wherein asurface area of the first working electrode and a surface area of thesecond working electrode are substantially the same.
 3. The methodaccording to claim 1, wherein a surface area of the first workingelectrode and a surface area of the second working electrode aredifferent, wherein step i) comprises: determining a current flowingbetween the first working electrode and the first counter electrode;determining a current flowing between the second working electrode andthe second counter electrode; normalizing the current flowing betweenthe first working electrode and the first counter electrode and thecurrent flowing between the second working electrode and the secondcounter electrode to a same electrode surface area to yield a normalizedcurrent flowing between the first working electrode and the firstcounter electrode and a normalized current flowing between the secondworking electrode and the second counter electrode; and subtracting thenormalized current flowing between the second working electrode and thesecond counter electrode from the normalized current flowing between thefirst working electrode and the first counter electrode, whereby acorrected current is obtained.
 4. The method according to claim 1wherein the first working electrode and the first counter electrode areseparated by less than about 500 μm.
 5. The method according to claim 1wherein the first working electrode and the first counter electrode areseparated by less than about 200 μm.
 6. The method according to claim 1wherein the second working electrode and the second counter electrodeare separated by more than about 500 μm.
 7. The method according toclaim 1 wherein the first working electrode and the second counterelectrode are separated by more than about 500 μm.
 8. The methodaccording to claim 1 wherein the second working electrode and the secondcounter electrode are separated by more than about 1 mm.
 9. The methodaccording to claim 1 wherein the first working electrode and the secondcounter electrode are separated by more than about 1 mm.
 10. The methodaccording to claim 1 wherein the redox species is a mediator and theconcentration of the reduced or oxidized form of the mediator isindicative of a concentration of an analyte and wherein a measure of adiffusion coefficient of a reduced or oxidized form of the mediator isdetermined as a precursor to the determination of the concentration ofthe analyte.
 11. The method according to claim 10 wherein the analyte isglucose.
 12. The method according to claim 1 wherein the redox speciesis a mediator.
 13. The method according to claim 1 wherein theelectrochemical cell additionally comprises a separate referenceelectrode.
 14. Apparatus for determining the concentration of a redoxspecies in an electrochemical cell comprising: an electrochemical cellhaving a first working electrode, a first counter electrode, a secondworking electrode and a second counter electrode, characterized in thatthe first working electrode is spaced from the first counter electrodeby less than 500 μm, the first working electrode is spaced from thesecond counter electrode by more than 500 μm, and the second workingelectrode is spaced from the second counter electrode by more than 500μm; an electrical circuit capable of applying an electric potentialdifference between the first working electrode and the first counterelectrode; and an electrical circuit capable of applying an electricpotential difference between the second working electrode and the secondcounter electrode.
 15. The apparatus according to claim 14, wherein theapparatus is a glucose meter.
 16. A method for determining aconcentration of a reduced or oxidized form of a redox species, themethod comprising the steps of: a) providing an electrochemical cellcomprising a first working electrode, a second working electrode, and acounter electrode; b) selecting a spacing between the first workingelectrode and the counter electrode such that reaction products from thecounter electrode arrive at the first working electrode; c) selecting aspacing between the second working electrode and the counter electrodesuch that a significant amount of reaction products from the counterelectrode do not arrive at the second working electrode; d) applying anelectric potential difference between the second working electrode andthe counter electrode whereby the second working electrode issubstantially charged and whereby surface group reactions aresubstantially completed; e) interrupting a circuit between the secondworking electrode and the counter electrode before a significant amountof the redox species is reacted at the second working electrode; f)applying an electric potential difference between the first workingelectrode and the counter electrode; g) selecting a potential of thefirst working electrode such that a rate of electro-oxidation of thereduced form of the redox species or a rate of electro-reduction of theoxidized form of the redox species is diffusion controlled; h)determining a current flowing between the first working electrode andthe counter electrode; and i) obtaining from the current a valueindicative of a concentration of the reduced form of the redox speciesor the oxidized form of the redox species.
 17. A method for determininga concentration of a reduced or oxidized form of a redox species, themethod comprising the steps of: a) providing an electrochemical cellcomprising a first working electrode, a second working electrode, and acounter electrode; b) selecting a spacing between the first workingelectrode and the counter electrode such that reaction products from thecounter electrode arrive at the first working electrode; c) selecting aspacing between the second working electrode and the counter electrodesuch that a significant amount of reaction products from the counterelectrode do not arrive at the second working electrode; d) applying anelectric potential difference between the second working electrode andthe counter electrode and between the first working electrode and thecounter electrode, whereby the second working electrode and firstworking electrode are substantially charged and whereby surface groupreactions are substantially completed; e) interrupting a circuit betweenthe second working electrode and the counter electrode before asignificant amount of the species is reacted at the second workingelectrode; f) applying an electric potential difference between thefirst working electrode and the counter electrode; g) selecting apotential of the first working electrode such that a rate ofelectro-oxidation of the reduced form of the redox species or a rate ofelectro-reduction of the oxidized form of the redox species is diffusioncontrolled; h) determining a current flowing between the first workingelectrode and the counter electrode; and i) obtaining from the current avalue indicative of a concentration of the reduced form of the redoxspecies or the oxidized form of the redox species.
 18. Apparatus fordetermining a concentration of a redox species in an electrochemicalcell comprising: an electrochemical cell having a first workingelectrode, a first counter electrode, a second working electrode and asecond counter electrode, characterized in that the first workingelectrode is spaced from the first counter electrode by less than 500μm, the first working electrode is spaced from the second counterelectrode by more than 500 μm, and the second working electrode isspaced from the second counter electrode by more than 500 μm; means forapplying an electric potential difference between the first workingelectrode and the first counter electrode; and means for applying anelectric potential difference between the second working electrode andthe second counter electrode.
 19. The apparatus according to claim 18,wherein the apparatus is a glucose meter.
 20. An electrochemical cellcomprising a first working electrode, a first counter electrode, asecond working electrode, and a second counter electrode, the firstworking electrode being spaced from the first counter electrode by lessthan about 500 μm, the first working electrode being spaced from thesecond counter electrode by more than about 500 μm, and the secondworking electrode facing the second counter electrode and being spacedfrom the second counter electrode by more than about 500 μm.
 21. Theelectrochemical cell according to claim 20, wherein the first workingelectrode and the first counter electrode are facing one another. 22.The electrochemical cell according to claim 20, wherein the firstworking electrode and the first counter electrode are in a side-by-sideconfiguration.
 23. The electrochemical cell according to claim 20,wherein the first working electrode and the second working electrode areof substantially corresponding area.
 24. The electrochemical cellaccording to claim 20, further comprising a separate referenceelectrode.
 25. The electrochemical cell of claim 20, wherein theelectrochemical cell is a hollow electrochemical cell.
 26. Theelectrochemical cell according to claim 25, having an effective cellvolume of less than 1.5 microliters.